Sixteen-membered macrolide antibiotics are highly safe and useful in clinical treatments and effective on, for example, Gram-positive bacteria, Mycoplasma and Chlamydia. Thus they have been employed worldwide in various fields including pediatrics. In addition, resistant to these antibiotics is scarcely induced and, compared with 14-membered macrolide, 16-membered macrolide antibiotics exert less interaction with other drugs and less affect on the intestinal tract. Further, they give little irritation (bitterness) at the oral administration. These characteristics make the 16-membered macrolide antibiotics excellent antimicrobial agents for improving the quality of life of patients. Among all, miokamycin (MOM) [Journal of Antibiotics, 29 (5), 536 (1976)] and rokitamycin (RKM) [Journal of Antibiotics, 34 (8), 1001 (1981)] have been frequently used clinically as a semisynthetic 16-membered macrolide antibiotic superior to natural compounds in the action of preventing infection. On the other hand, studies on derivatives of these compounds have been vigorously performed in order to improve the efficacy and safety thereof.
The present inventors conducted biochemical studies on 16-membered macrolide derivatives effective on various Gram-positive bacteria and found so far two fungi capable of specifically removing an acyl group binding to a hydroxyl group at the 3-position of a lactone ring of a 16-membered macrolide (EP-A-526,906 and U.S. Pat. No. 5,219,736). On the other hand, similar biochemical reactions effected by Bacillus subtilis have been already reported [Journal of Fermentation Technology, 57 (6), 519 (1979)].
Subsequently, the present inventors paid their attention to the drug metabolism in MOM and RKM which might be called "16-membered macrolide antibiotics of the second generation". Namely, two acyl groups in the neutral sugar moiety in these antibiotics are removed with esterase in vivo and the neutral sugar moiety is metabolized into mycarose having two free hydroxyl groups. It is reported that the antimicrobial activities in vitro of these metabolites are thus reduced [Yakugaku Zasshi, 102 (8), 781 (1982), Symposium on novel drugs IV, TMS-19-Q, p. 109, 119, 31st General Meeting of Society of Japan Chemotherapy]. Through feedback of these findings to drug design, the present inventors conducted repeated studies on the chemical synthesis of 16 -membered macrolide derivatives being stable in vivo and retaining antimicrobial activity even after being metabolized. As a result, they succeeded in the synthesis of a novel derivative, wherein both of two hydroxyl groups in the mycarose moiety of a 16-membered macrolide derivative form ether bonds with alkyl groups, and proved that said compound showed a remarkable long-acting in vitro (EP-A-560,147).
In order to improve the antimicrobial activity and/or pharmacokinetics of 16-membered macrolides, a number of derivatives thereof have been synthesized by partly acylating the hydroxyl groups therein. On the other hand, several groups of workers have already reported the synthesis of derivatives by monoalkylating hydroxyl group(s) in the mycarose moiety of 16-membered macrolides [Chemistry Letters, 769 (1977); JP-A-60-58998; and JP-A-62-234093; the term "JP-A" as used herein means an "unexamined published Japanese patent application"].
First, the present inventors discussed effects of a difference in the structure at the 9-position among midecamycin analogues, from among 16-membered macrolide compounds, on pharmacokinetics. That is to say, midecamycin A.sub.1 having a hydroxyl group at the 9-position (Medemycin; MDM) [Journal of Antibiotics, 24 (7), 452 (1971)] and midecamycin A.sub.3 having a carbonyl group at the 9-position [ibid., 24 (7), 476 (1971)] were orally administered to animals (mice), followed by observing the pharmacokinetics. As a result, it was confirmed that MDM having a hydroxyl group at the 9-position was superior to midecamycin A.sub.3 both in the sustained concentration in serum and in the recovery in urine. Based on these results, it has been judged that a 16-membered macrolide derivative, in particular, a midecamycin analogue having a hydroxyl group at the 9-position is preferred to the one having a carbonyl group at the same position for the improvement in the pharmacokinetics. It has been also reported that, regarding the 13-position of 16-membered macrolides belonging to iso-forms, a derivative having a hydroxyl group is superior to the one having a carbonyl group in the efficacy in vivo [Scientific Reports of Meiji Seika Kaisha, 13, 100 (1973)].
In order to clarify the correlation among structures of 16-membered macrolide compounds, to study biosynthesis of these compounds and to analyze the structures thereof, there have been known methods for reducing a carbonyl group at the 9-position of a 16-membered macrolide compound into a hydroxyl group through a synthetic chemical approach [for example, those described in Journal of Organic Chemistry, 39 (16), 2474 (1974); Journal of Antibiotics, 34 (12), 1577 (1981); and ibid., 39 (12), 1784 (1986)] and through a biochemical approach [for example, those described in JP-A-50-126880; Journal of Antibiotics, 32 (7), 777 (1979); and ibid., 33 (8), 911 (1980)].
The present inventors then paid their attention to the 3-position of a lactone ring of 16-membered macrolide derivatives. Regarding the 3-position of 16-membered macrolide compounds, in particular, leucomycin analogues, correlations among the structures and various activities including pharmacokinetics have been studied in detail [Journal of Antibiotics, 21 (9), 532 (1968)]. At the early stage, it was reported that a compound having a free hydroxyl group at the 3-position of a lactone ring was superior to the corresponding 3-O-acyl compound in the antimicrobial activity in vitro but showed a low concentration in serum in vivo [Hakko to Kogyo, 37 (12), 27 (1979)]. However, subsequent studies have revealed that a certain derivative having a modified neutral sugar moiety sustains a strong antimicrobial activity, even though having a free hydroxyl group at the 3-position, and achieves a high concentration in serum when orally administered to animals of small or middle size [Journal of Antibiotics, 34 (8), 1001, (1981)] . Therefore techniques for removing an acyl group directly binding to a hydroxyl group at the 3-position of a lactone ring have attracted public attention again as a method for enhancing the antimicrobial activity in vitro of a 16-membered macrolide derivative.
The present inventors have recently synthesized novel 16-membered macrolide derivatives having two ether bonds in the mycarose moiety, for example, 4"-O-depropionyl-4"-O-isoamyl-3"-O-methylmidecamycin A.sub.3 (EP-A-560,147). Compared with 16-membered macrolide antibiotics put into the market in recent years, these novel 16-membered macrolide derivatives are clearly improved in the maximum concentration in serum and the recovery in urine in animal experiments with the use of mice because the mycarose moiety of these compounds is scarcely attacked by esterase. However, these 16-membered macrolide derivatives are not always satisfactory in the pharmacokinetics. The existence of a carbonyl group at the 9-position, as described above, is considered as one of the factors to be solved for obtaining excellent pharmacokinetics. Thus there has been first required to develop a novel 16-membered macrolide derivative which is stable in vivo, scarcely reduces the antimicrobial activity even after being metabolized and is superior to the derivatives having a carbonyl group at the 9-position and two ether bonds in the mycarose moiety in the pharmacokinetics.
The novel 16-membered macrolide derivative having two ether bonds in the mycarose moiety, for example, 4"-O-depropionyl-4"-O-isoamyl-3-O-methylmidecamycin A.sub.3 has enhanced antimicrobial activity on some bacteria belonging to the genus Streptococcus or Branhamella, compared with MOM. However, it is still desired to improve its antimicrobial activity in vitro. By the way, it cannot be expected that the antimicrobial activity in vitro is remarkably improved by reducing the carbonyl group at the 9-position into a hydroxyl group. Thus it is proposed to remove a propionyl group binding to a hydroxyl group at the 3-position of a lactone ring, as described above, as a method for exerting an excellent antimicrobial activity in vitro without considerably enhancing a certain toxicity, such as acute toxicity, inherent to the compound per se. That is to say, it is secondly required to develop a novel 16-membered macrolide derivative which is stable in vivo, scarcely reduces the antimicrobial activity even after being metabolized, shows excellent pharmacokinetics as much as possible and is superior to the novel 16-membered macrolide derivative prepared by the present inventors so far in antimicrobial activity in vitro.
Under these circumstances, it has been required to develop a novel macrolide derivative wherein both of two hydroxyl groups in the mycarose moiety form ether bonds with alkyl groups and, at the same time, an sp.sup.3 carbon is located at the 9-position. In fact., there has never been reported such a derivative, either the hydroxyl group at the 3-position of a lactone ring is an acylated hydroxyl group or a free hydroxyl group.
In order to prepare such derivatives, in particular, those having a free hydroxyl group at the 3-position of a lactone ring, in practice, it is necessary to perform a chemical reaction consisting of a plural number of steps (EP-A-560,147) involving regio- and stereo-selective glycosylation for introducing a neutral sugar and two continuous steps of microbial conversion. Therefore, the cost and time for preparing these derivatives are not always completely satisfactory. Furthermore, an activator, which is dangerous and to be handled with care, should be stoichiometrically used in the above-mentioned glycosylation and there are some problems in scaling up thereof. Accordingly, it has been thirdly required to establish a process for producing a 16-membered macrolide derivative, wherein two hydroxyl groups in the mycarose moiety form ether bonds with alkyl groups, by chemical synthesis without using any glycosylation.
In the chemical modification of a 16-membered macrolide antibiotic, there have been known some cases wherein an alkyl group is introduced into the secondary hydroxyl group at the 4"-position of the mycarose moiety via no glycosylation. For example, Omura, Sano et al. introduced an alkyl group into the secondary hydroxyl group at the 4"-position of the mycarose moiety by protecting a hemiacetal hydroxyl group formed at the 3,18-position of spiramycin I (JP-A-60-58998; and JP-A-60-239494). On the other hand, Yoshioka et al. modified two hydroxyl groups in the mycarose moiety of a 16-membered macrolide derivative with dialkyl tin and introduced, for example, a benzyl group to the secondary hydroxyl group, which was one of the two hydroxyl groups, at the 4"-position (JP-A-62-234093). However, there has never been reported so far a reaction whereby an alkyl group is introduced into the tertiary hydroxyl group at the 3"-position of the mycarose moiety of a 16-membered macrolide derivative via no glycosylation.